4 We have shown that menthol attenuates both capsaicin and NKA-induced ... capsaicin-induced bronchoconstriction could be, in part, explained by a direct ...
British Journal of Pharmacology (1997) 121, 1645 ± 1650
1997 Stockton Press
All rights reserved 0007 ± 1188/97 $12.00
Capsaicin and neurokinin A-induced bronchoconstriction in the anaesthetised guinea-pig: evidence for a direct action of menthol on isolated bronchial smooth muscle C.E. Wright, E.A. Laude, T.J. Grattan & A.H. Morice Section of Respiratory Medicine, Department of Medicine and Pharmacology, University of Sheeld, Beech Hill Rd., Sheeld S10 2RX 1 For many years menthol has been used in the treatment of respiratory disorders although, a bronchodilator eect of menthol has yet to be described. Using the bronchoconstrictors capsaicin (acting via stimulating the release of neuropeptides from sensory aerents) and neurokinin A (NKA) we have raised airways resistance in the guinea-pig (GP) and studied the eect of menthol on both capsaicin and NKA-induced bronchoconstriction in vivo. In vitro the eect of menthol on acetylcholine (ACh) and KCl precontracted GP bronchi was also studied. 2 GP (n=13) were anaesthetized (urethane 1.5 g kg71, i.p.) and a bolus injection of capsaicin (7.5 mg ml71, i.v.) or infusion of NKA (1 mg min71, i.v.) was given either in the presence of air (0.81 min71) or air impregnated with menthol vapour (7.5 mg l71) freely breathed from a tracheal cannula via a T-piece. Airways resistance (Raw) and ventilation were measured throughout. Bronchi of mean internal diameter (1029+73.6 mm; n=24) were removed from GP (n=16) and mounted in the Cambustion myograph. Bronchial rings were maximally precontracted with 80 mM KC1 or 2 mM ACh. Relaxation due to a cumulative dose of menthol (1 ± 3000 mM) was measured. 3 Menthol produced a signi®cant (P50.05) 51.3% reversal of the capsaicin-induced increase in Raw, and also inhibited the signi®cant (P50.05) reduction in minute ventilation (Ve) associated with the capsaicin-induced increased in Raw. Menthol also caused a signi®cant (P50.05) 41% reversal of the NKA-induced increase in Raw. The NKA-induced decrease in Ve was again signi®cantly (P50.05) reversed with menthol inhalation. Menthol caused a signi®cant (P50.001) dose-dependent relaxation of KCl and ACh precontracted bronchi. 4 We have shown that menthol attenuates both capsaicin and NKA-induced bronchoconstriction in vivo and relaxes KCl and ACh preconstricted bronchi in vitro. Menthol inhibition of NKA and capsaicin-induced bronchoconstriction could be, in part, explained by a direct action of menthol on bronchial smooth muscle. Keywords: Menthol; capsaicin; neurokinin A; airways resistance; bronchodilatation
Introduction Menthol has been used for many years in a wide range of over the counter medications. As a medicine, its most popular application is in the relief of common cold symptoms such as cough and chest congestion, although there is very little objective clinical evidence to show that menthol has any bene®cial eects at the levels used in proprietary cough and cold products. Antitussive activity of menthol has recently been demonstrated in both healthy volunteers (Morice et al., 1994) and in guinea-pigs (Laude et al., 1994) with the citric acid-induced cough model. It was suggested by Laude that the antitussive properties of menthol in the guinea-pig may be due to a direct inhibitory action on the cough re¯ex, although an action on other respiratory mechanisms associated with cough, such as bronchoconstriction and airways secretion could not be ruled out. Evidence of an eect of menthol on pulmonary function is very limited. Cohen & Dressler (1982) have studied the eects of a mixture of aromatic vapours (including menthol) on the calibre of airways in volunteers suering from the common cold. By measuring forced expiratory volumes, peak expiratory ¯ow rate and lower as well as total airways resistance, an improvement in airway's calibre with a 20 ± 60 min aromatic vapour inhalation was found. However, since a mixture of aromatic vapours was used it is hard to attribute any eects of the mixture to menthol alone. Tamaoki et al. (1995) have more recently demonstrated a reduction in airway hyperresponsiveness, measured as a shift in the methacholine dose-response curve, in patients with mild asthma following long-term treatment with menthol. However, this group did not ®nd any
improvement in measures of forced expiratory volume in 1 s (FEV1) following menthol treatment, which suggests that menthol had no eect on airway's calibre in these patients. Thus evidence of bronchodilatation following menthol inhalation has yet to be obtained, furthermore there is no evidence of a direct action of menthol on bronchial smooth muscle. However, menthol has been shown to relax ileal smooth muscle in guinea-pigs by Hawthorn et al. (1988) and a speci®c action of menthol on airways epithelium has been demonstrated by Chiyotani et al. (1994), in dog, where menthol application in vitro increased Cl7 secretion. In the present study, we have examined the eect of prior menthol inhalation on caspsaicin-induced increase in airways resistance (Raw) in the guinea-pig. In view of the complex mechanism whereby capsaicin induced bronchoconstriction, involving sensory nerve stimulation and neuropeptide release, further studies have been designed to investigate the possible sites of action of menthol by use of exogenous neurokinin A in vivo and in preparations of isolated bronchi preconstricted with potassium chloride (KCl) and acetylcholine (ACh).
Methods Airways resistance Airways resistance was determined in Dunkin-Hartley guineapigs (420 ± 645 g; n=13) anaesthetized with intraperitoneal
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C.E. Wright et al
urethane (1.5 g kg71) as previously described (Bee et al., 1995). Blood pressure was continuously monitored from the carotid artery (Viggo-Spectromed pressure transducer with Lectromed ampli®cation). Intravenous infusions or bolus injections were administered via a jugular vein cannula. Pleural pressure was measured by introducing a cannula connected to a pressure transducer, into . the pleural cavity between the ®fth and sixth ribs. Air¯ow (V) was measured from a tracheal cannula which was connected to a pneumotachograph (Fleisch 0.6 V) and volume (V) was determined from the integrated ¯ow. The animals were allowed to breathe spontaneously from an air¯ow of 0.8 l min71 passed across the tracheal cannula via a Tpiece. Airways resistance was measured from the pleural pressure-tracheal air¯ow relationship. A voltage which was proportional to lung volume was electronically subtracted from the pleural pressure and the resulting signal fed into one axis of an X-Y pen recorder, while the air¯ow signal was fed into the other axis. Signals were in phase. Total airways resistance (Raw) was calculated from the slope of the pressure-¯ow loop.
Menthol Menthol (Sigma Chemicals, Poole, Dorset U.K.) vapours were administered to the animal by passing air at 0.8 l min71 through a glass tube containing a cotton wool plug impregnated with 2.5 g of menthol crystals. The animal was allowed to freely breath the aromatic vapours, from the tracheal cannula via a T-piece. The concentrtion of menthol delivered to the animal with this technique was (7.5 mg l71) at a rate of 0.8 l min71. The concentration of menthol administered was quanti®ed by purging the output air through a dreschel bottle containing isopropyl alcohol. Analysis of the resulting solution was by gas chromatography Fisons GC-MS (GC8000 & MD800) with selected ion monitoring for menthol (Laude et al., 1994).
Capsaicin Repeated doses of capsaicin (7.5 mg ml71, i.v.) were given as 400 ml bolus injections via the jugular vein. Before the injection of capsaicin, air (0.8 l min71) or exposure to air impregnated with menthol (7.5 mg l71) was freely breathed by the animal for 5 min, the delivery of the air or menthol was promptly ceased post-injection. Each capsaicin response was monitored for 420 min, with both Ve and Raw allowed to return to basal levels between injections.
NKA Because of the rapid metabolism of neuropeptides in vivo and the short duration of bolus injections, 1 mg min71 NKA was administered as an infusion. Post 20 min infusion, either air (0.8 l min71) or air impregnated with menthol (7.5 mg l71) was administered for 5 min. Values of Raw before, during and after air or menthol delivery were compared.
Myography Male Dunkin-Hartley guinea-pigs (400 ± 750 g, n=16) were anaesthetized with intraperitoneal urethane (1.5 g kg71) and the lungs removed. Bronchi (n=24) of internal diameter (1029+73.6 mm), were dissected free of any connective tissue and mounted on two 40 mm wires. The eects of menthol upon bronchi preconstricted with KCl and ACh were studied in a myograph (Cambustion Biological, Cambridge) at a calculated transmural pressure of 20 mmHg, which in preliminary experiments had been shown to give the maximum contractile response to 80 mM KCl. The bronchi were bathed in a physiological saline solution (PSS), pH 7.4 of composition (in mM): NaCl 120, KCl 4.7, CaCl2.2H2O 2.5, MgSO4.7H2O 1.17,
Menthol and induced bronchoconstriction
NaHCO3 35, KH2PO4 1.18, EDTA 0.269 and glucose 5.5. The 5 ml organ bath was aerated with a 95% O2/5% CO2 gas mixture and the temperature held at 378C. Bronchi were allowed to equilibrate for 30 ± 40 min before the experimental procedures were commenced. At the start of each experimental procedure 80 mM KCl was applied on at least three successive occasions or until the response was reproducible. The eect of menthol was then studied on bronchi precontracted with KCl (80 mM) or ACh (2 mM) by use of cumulative concentrations of menthol within the range 1 ± 3000 mM. Each concentration was allowed to equilibrate for 15 min, by which time a maximum sustained relaxation was attained. Control solutions of menthol vehicle (ethanol 12% in saline) were added to both ACh and KCl precontracted bronchial rings. Smooth muscle relaxation associated with menthol application had an onset of action of between 5 ± 10 min. Preliminary experiments showed that the dilatation induced by menthol could be maintained for between 10 ± 15 min if the organ bath was left uncovered and for 35 ± 40 min if covered. The dierence in duration was probably due to loss of this volatile terpine alcohol to the atmosphere and thus subsequent experiments were carried out with the bath covered.
Chemicals Menthol, NKA and ACh were obtained from Sigma Chemicals (Poole, Dorset, U.K.). Menthol was dissolved in ethanol (20%) and subsequently diluted with saline to give a stock solution of 100 mM. NKA, ACh and KCl were dissolved in sterile, physiological saline. All other reagents were obtained from BDH Chemicals Ltd (Lutterworth Lecs, U.K.).
Statistics Results are expressed as mean (+s.e.mean). For comparison of airways resistance response paired t tests were used, calculation of regression lines and r values were by Microsoft Excel 5.0. For myography, IC50 values were calculated by use of Gra®t, plotting data as a sigmoid curve. Results were tested for signi®cance by analysis of variance-single ANOVA. Values of P50.05 were considered signi®cant.
Results Airways resistance Baseline Raw Menthol inhalation (5 min) had no signi®cant eect upon basal Raw. Calculated basal Raw with and without menthol inhalation were 2.64+0.27 and 2.63+0.23 mmHg min71, respectively. Capsaicin Capsaicin (bolus injection, i.v.) consistently produced an increase in airways resistance which stabilized after 10 min. There was a within animal variation in sensitivity to capsaicin with air pretreatment (0.8 l min71) giving maximal increases in Raw from baseline ranging from 9.3 to 57.9 mmHg min71. Capsaicin injection with air pretreatment gave a mean increase in Raw from 9.1+2.6 to 29.0+4.8 mmHg min71, whereas prior menthol inhalation gave a mean increase in Raw from 8.4+2.2 to 17.9+3.9 mmHg min71, a statistically signi®cant (51.3%, P50.01) inhibition of the capsaicin-induced increase in Raw (Figure 1). In several animals, successive injections of capsaicin caused a potentiation of the maximal Raw response. However, this did not prove signi®cant on analysis of the whole group. NKA NKA (infusion, i.v.) 1 mg min71 consistently caused an increase in airways resistance, which attained 80% of maximum (15.43+1.8 mmHg min71) after 20 min. However,
Prior air+Caps
Prior menthol Prior air+Caps +Caps
Figure 1 Increase in airways resistance (Raw; mean+s.e.mean) following a 400 ml injection of capsaicin (Caps; 7.5 mg ml71, bolus i.v.) pretreated with either 5 min tracheal air administration (0.8 l min71), or 5 min air plus menthol vapour (7.5 mg l71). There was a signi®cant dierence between air and menthol pretreatment groups; *P50.01.
subsequently there was a slow progressive potentiation of the NKA-induced increase in Raw during the course of the experiment, increasing from 12.07+1.0 ± 15.43+1.8 mmHg min71. The maximum increase in Raw from basal levels with NKA infusion over 50 min varied between animals (range 2.96 ± 29.52 mmHg min71). Air inhalation during NKA infusion had little eect upon the NKA-induced increase in Raw 9.48+2.4 to 9.84+2.3. In contrast, menthol inhalation caused a statistically signi®cant (41.3%, P50.01) reversal in the NKA-induced increase in Raw from 12.6+3.2 to 7.37+3.0 (Figure 2), which was still evident 5 min after the initial exposure to menthol but returned to raised basal levels within 10 min.
Ventilation Capsaicin bolus injection Menthol inhalation had no signi®cant eect on basal tidal volume (Vt) or frequency. Bolus intravenous injection of capsaicin with prior inhalation of air produced a signi®cant (42.5%, P50.01) decrease in minute ventilation corrected for 100 g body weight (VeBt). The reduction in VeBt varied markedly between animals (range 0.45 ± 15.1 ml min71 100 g71) and was mostly due to a signi®cant (P50.001) fall in Vt from 2.42+0.20 to 1.6+0.18 ml and to a lesser extent a non-signi®cant decrease in respiratory rate from 57.1+14.0 to 42.7+11.2 breaths min71. The capsaicin-induced changes in Raw with air pretreatment correlated (r2=0.71) closely with the changes in VeBt. Inhalation of menthol, 5 min before capsaicin injection, prevented the capsaicin induced decrease in VeBt. Mean values of VeBt post-capsaicin were not signi®cantly dierent from basal values pre-capsaicin (Figure 3). Approximately 30 min after menthol inhalation, a further capsaicin injection with prior air inhalation again signi®cantly (36.4%, P50.01) reduced VeBt, which was not signi®cantly dierent from that induced by the initial capsaicin injection (Table 1). NKA infusion NKA-induced increase in Raw demonstrated a close correlation (r2=0.80) with the reduction in VeBt. The maximum reduction in VeBt from basal again varied between animals (range 0.83 ± 24.3 ml min71 100 g71). A 5 min inhalation of air (0.8 l min71) during the infusion had no effect upon the NKA-induced decrease in VeBt. However, a 5 min inhalation of menthol vapour (7.5 mg l71) caused a signi®cant (40%, P50.01) reversal of the NKA-induced decrease in VeBt (Figure 4) correlating closely with changes in Raw. This improvement in VeBt was due to a statistically signi®cant (P50.01) increase in both Vt and respiratory rate (Table 1).
8 6 NKA+air
5
*
10
NKA
10
12
NKA
*
15
14
NKA+menthol
20
16
NKA
25
18
NKA+air
30
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20
NKA
35
Mean increase in Raw (mmHg min–1)
40
Menthol and induced bronchoconstriction
Figure 2 Increase in airways resistance (Raw; mean+s.e.mean) following a neurokinin A (NKA) infusion (1 mg min71) with treatments of either 5 min tracheal air administration (0.8 l min71), or 5 min plus menthol vapour (7.5 mg l 71). There was a signi®cant dierence between air and menthol pretreatment, *P50.01.
Mean decrease in Ve (ml min–1 100g–1)
Mean increase in Raw (mmHg min–1)
C.E. Wright et al
12 10 8 6 4 2
*
0 –2 –4 –6 Prior air+Caps Prior menthol +Caps
Prior air+Caps
Figure 3 Decrease in minute ventilation corrected for 100 g body weight (VeBt; mean+s.e.mean) following a 400 ml injection of capsaicin (Caps; 7.5 mg ml71, bolus i.v.) pretreated with either 5 min tracheal air administration (0.8 l min71), or 5 min air plus menthol vapour (7.5 mg l71). There was a signi®cant dierence between air and menthol pretreatment groups; *P50.01.
Table 1 A summary of the eect of menthol and air on capsaicin and NKA-induced changes in airway resistance and ventilation Raw Caps with Caps with NKA and NKA and
prior air prior menthol air menthol
424 **170 507 **302
Decrease/increase (%) Frequency VT ±14.6 *9.5 ±25.4 *±15.1
±33.5 *8.9 34.9 *75.0
Ve ±41.2 *23.8 ±3.14 *38.9
Results are expressed as % decrease (-ve values) or increase (+ve values) from baseline. Signi®cance was calculated from mean absolute dierence between control air and menthol inhalation by use of paired t test. *P
